Amide bonds assemble pili on the surface of bacilli.

Department of Microbiology, University of Chicago, Chicago, IL 60637, USA.

Abstract

Pilin precursors are the building blocks of pili on the surface of Gram-positive bacteria; however, the assembly mechanisms of these adhesive fibers are unknown. Here, we describe the chemical bonds that assemble BcpA pilin subunits on the surface of Bacillus cereus. Sortase D cleaves BcpA precursor between the threonine (T) and the glycine (G) residues of its LPXTG sorting signal and catalyzes formation of an amide bond between threonine (T) of the sorting signal and lysine (K) in the YPKN motif of another BcpA subunit. Three CNA B domains of BcpA generate intramolecular amide bonds, and one of these contributes also to pilus formation. Conservation of catalysts and structural elements in pilin precursors in Gram-positive bacteria suggests a universal mechanism of fiber assembly.

Inter- and intramolecular amide bonds in BcpA pili. (A) RP-HPLC of CNBr cleaved and Ni-NTA purified BcpA pili. Compounds A-C were analyzed by SDS/PAGE (Coomassie), by immunoblotting with α-BcpA and by binding to His-HRP. (B) The molecular mass of compound A was determined by ESI-FTMS. Insets display the deduced structure of compound A and the Mr values calculated by XtractAll for Qual Browser. Intra- (Asn163-Lys37, black), intermolecular amide (Lys162-Thr522, red), YPKN motif (blue) and LPXTG sorting signal (red) are indicated. M* denotes homoserine lactone residues. Residues in bold were identified by Edman degradation. (C) Compound A was incubated with trypsin and analyzed by SDS/PAGE (Coomassie). Compound A1, a tryptic peptide of compound A, was purified by Ni-NTA affinity chromatography, separated by RP-HPLC (arrowhead) and mass measured by MALDI-MS. (D) CAD fragmentation spectra (m/z) of the parent ion 1,153.594+. Fragment ions are labeled,‘ refers to fragment ions that arose from fragmentation of the GTLTIHKYEQEK branch of the peptide, “ refers to fragment ions that arose from the LPVT branch of the peptide. Unmarked fragment ions were derived from fragmentation of the HHHGAVLNYDVHLYPKNEIM*. M* denotes a homoseryl residue.

Cleavage sites of sortases and their contribution to pilus assembly. (A) Diagram displays the precursor (P1) of BcpA-GST, and the signal peptidase (P2) and sortase cleaved products (M). (B) Pilus formation from BcpA-GST substrate was examined by immunoblotting with α-BcpA in cells harboring both sortase D (SrtD) and sortase A (SrtA), only one of the two sortases or none at all. (C) Bacilli were analyzed by ImmunoGold labeling with α-BcpA serum and viewed by transmission electron microscopy. (Scale bars, 1 μm.) (D) The cell wall of bacilli was removed with PlyL, protoplast lysed by sonication, and sortase cleavage products purified by affinity chromatography on glutathione-Sepharose. Electrophoretic mobility of the molecular weight marker, pilin precursors (P1/P2), and mature cleavage products (M) on SDS/PAGE is indicated. Edman degradation revealed the N-terminal sequences of mature products. (E) Purified cleavage products were subjected to immunoblotting with specific antisera (α-GST or α-BcpA).